The European patent application with the publication number (EP-A) 0,161,629, and South African Patent 85/3672 disclose the use of the DNA coding for the signal peptide (prepeptide) of the .alpha.-amylase inhibitor tendamistat in order for a Streptomycetes cell to excrete a polypeptide, in particular tendamistat. In this regard, South African Patent No. 85/3672 at page 2, lines 24-30, teaches that the signal peptide of tendamistat is Met-Arg-Val-Arg-Ala-Leu-Arg-X-Ala-Ser-Ala in which X represents a hydrophobic region comprising 10 to 25, preferably 17 to 20 amino acids (most likely 20 amino acids) (SEQ ID NO:48). The appropriate DNA can, in principle, be obtained from every strain producing tendamistat, but a DNA obtained as in Example 3 of German Offenlegungsschrift 3,331,860 is preferably used. German Patent Application P 37 07 150.5, filed Mar. 6, 1987, has already proposed a process for the excretion of fusion proteins from Streptomycetes, which comprises incorporating the coding sequence, which has been modified where appropriate, and expressing the recombinant gene in a Streptomycetes cell. Thus, in this case the tendamistat structural gene is used as a "carrier" for another gene, the fusion proteins which are obtained having the amino acid sequence of another protein located within the tendamistat amino acid sequence. Consequently, on chemical or enzymatic cleavage of this fusion protein to liberate the other protein, two tendamistat part-sequences are obtained. Said German patent application P 37 07 150.5 also relates to tendamistat derivatives, which are understood to include those with a markedly shortened amino acid chain. Derivatives of this type are able in a reversible manner to react with the specific receptors in the form of a competitive inhibitory mechanism.
In the European patent application with the publication number (EP-A) 0,289,936 which corresponds to the German patent application DE 37 14 866 A1, now issued as German patent P 37 14 866.4, the inventors of the present application disclose the production of fusion proteins by coupling the structural gene for the desired protein to the 3'-end of the coding strand of the optionally modified tendamistat gene, expressing this genetic structure in a streptomyces host cell and isolating the secreted fusion protein from the supernatant. In a preferred embodiment the tendamistat gene is truncated at the 3'-end. For the truncation, the cleavage sites for the restriction enzyme BstEII in the region of triplets 31 and 32, StuI in the region of triplets 43 and 44, and Sau3A in the region of triplets 52 and 53 are used.
The present inventors have found that foreign proteins can also be prepared in Streptomycetes by constructing fusion protein genes in which the structural gene for the desired protein is coupled in the 3' end (of the coding strand) of the tendamistat gene, which has been modified where appropriate. The modification of the tendamistat gene may comprise, in particular, C-terminal shortening.
The DNA coding for tendamistat is depicted in EP-A 0,161,629 (where it is DNA sequence C (Table 1 in the annex of the present application)) (SEQ ID NO: 40). This structural gene contains several cleavage sites for restriction enzymes, which can be used to modify the coded amino acid sequence. Suitable cleavage sites are those for BstEII in the region of triplets 31 and 32, StuI in the region of triplets 43 and 44, and Sau3A in the region of triplets 52 and 53. It is possible, by incorporation of appropriate linkers, to insert at these sites one or more additional amino acids, to eliminate DNA segments between these cleavage sites, or to code for shortened amino acid sequences by incorporation of stop codons. Furthermore, it is possible by site-specific mutagenesis for any desired amino acids to be inserted, replaced or eliminated. In this way proteins are obtained which have an .alpha.-amylase inhibitory action, as well as proteins which do not have this activity but still react with the corresponding receptors.
The invention also relates to appropriate gene structures, vectors containing these gene structures, Streptomycetes cells transformed with these vectors, the excreted fusion proteins, and their use for the preparation of the foreign proteins and tendamistat derivatives.
In a further embodiment of the present invention, the present inventors have found that the process of the present invention can be used particularly well to prepare a fusion protein in which tendamistat portion is followed by a shortened proinsulin whose C chain comprises only one or two lysine residues ("mini-proinsulin"). These precursors can be converted particularly straightforwardly and economically into human insulin. Further embodiments of the invention include truncating the tendamistat portion, too, in fusion proteins of this type (EP-A 0,367,163 published on May 9, 1990).
Further embodiments of the present invention relate to advantageous gene structures and processes for the amplification and expression of the gene which codes for the fusion protein.
Surprisingly, the present inventors have discovered that fusion proteins with a very short tendamistat portion are stable in Streptomyces cells and are secreted into the medium. The fusion proteins obtained in this way behave like "mature" proteins because of the very short tendamistat chain. The present inventors have also discovered that fusion proteins containing a tendamistat portion and a C-terminal portion of a proinsulin derivative in which the B chain is connected to the A chain via a bridging member comprising Lys or Lys-Lys are, in fact, unexpectedly stable and are secreted into the medium, from which they can be isolated in high yields. Surprisingly, tendamistat mini-proinsulin derivatives characterized by an unnaturally short C-peptide are always secreted with correctly established disulfide bonds whereas tendamistat fusion proteins containing a authentic proinsulin moiety are secreted with incorrect disulfide linkages. This unique feature of tendamistat "mini-proinsulin" derivatives allows easy enzymatic cleavage to yield human insulin derivatives.